Modulated immunodominance therapy

ABSTRACT

The invention involves generating a T cell response to subdominant antigens and using the cells to therapeutically change the cellular homeostasis and nature of the immune response. In a preferred embodiment, the cells are generated outside of the patient avoiding the influence of the patient&#39;s immunologic milieu. By stimulating and growing the T cells from a patient in a tissue culture to one or more subdominant antigens and the transplanting them into the patient, if enough cells are expanded and transplanted, the transplanted cells overwhelm the endogenous dominant T cells in the response to either break or induce immune tolerance or otherwise modify the immune response to the cells or organism expressing that antigen. When the memory cells are established they are then reflective of this new immunodominance hierarchy so that the desired therapeutic effect is long lasting. In effect, the transplantation exogenously generated T cells reactive to the subdominant antigens is recapitulating priming and rebalancing the patient&#39;s immune response to target previously subdominant antigens in the cells or organism to produce a therapeutic benefit.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 14/122,036, entitled“Modulated Immunodominance Therapy,” filed Nov. 25, 2013, which is anational phase under 35 U.S.C. §371 of International Patent ApplicationNo. PCT/US12/39605, filed May 25, 2012, which claims priority to and thebenefit of U.S. Provisional Patent Application No. 61/490,505 entitled“Modulated Immunodominance Therapy,” filed May 26, 2011, the contents ofeach of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a novel therapeutic for cancer, chronicinfections, autoimmune diseases, and transplantation based upon themodification of the immunodominance hierarch by therapeutic manipulationof cellular homeostasis.

The clinical management of cancer is specific for each site of originand, for the most part, depends upon stage of the disease (i.e., how farthe tumor has invaded locally or spread to other organs by metastasis).Surgery and/or localized radiotherapy are generally the treatment ofchoice for the primary tumor with chemotherapy, monoclonal antibody orcytokine therapy or whole body irradiation being treatment formetastatic disease. Recently, the first dendritic cell therapy,Provenge, was approved for Prostate cancer with a 4-month progressionbenefit. Diagnosis is still based upon histologic analysis of thebiopsy. Molecular markers are sometimes standard if they will be helpfulin the selection of a drug (e.g., Herceptin). However, creating aprofile of the immune response has not been done on a routine clinicalbasis.

The environment in which a T cell sees an antigen during the primaryimmune response determines the nature of subsequent recall response. Theinitial recognition event and microenvironment around that primary cellcan result in many outcomes. If the antigen is presented by anonprofessional APC, only a subset of the epitopes may be releasedduring processing. If a costimulatory signal such as CD28 or TNF ismissing, the T cells may be anergized. Depending on themicroenvironment, the T cell may differentiate into a regulatory T cell,a T helper cell secreting Th1 cytokine (driving more of a cellularimmune response with proliferation of CD8⁺ CTL effectors) or a T helpercell secreting Th2 cytokine (driving more of a humoral immune responsewith proliferation and maturation of B cells and antibody production).In addition, the immune response will evolve such that T cellsresponding to certain epitopes of an antigen or certain antigens in themilieu will grow at the expense of T cells in the population which arereactive with other epitopes or antigens in the milieu. Because ofexponential cell growth, as the primary immune response subsides, theratio of these T cells is further accentuated and stored in the form ofmemory such that upon secondary stimulation, the immune response withinthat individual is focused on a small subset of the possible epitopes.While there are multiple mechanisms at work, as an operative model, theT cells that grow out to dominate the population are those responding tothe epitopes or antigens which come to dominate the immune response. Inthe primary immune response, they are growing out at the expense of theT cells responding to subdominant epitopes and, due to memory, dominatesubsequent immune responses.

Over a period of days after a person's immune system first sees anantigen, a dominant population of T cells responding to a limited numberof dominant epitopes is generated and these T cells determine the natureof the response to that antigen thereafter. While there are multipletypes of cells involved, the working model associated with thisinvention is that if the T cells responding to the dominant epitope onthe dominant antigens grow out as responsive T cells (e.g. CD4⁺:TH1,TH2, Treg, T follicular helper, TH17, TH22, TH9; CD8⁺ CTL's), a cellularor humoral immune response results. However, if the T cells in thedominant population are suppressive T cells (e.g., Treg, TH17, anergizedT cells), tolerance is induced. T cells responding to subdominantantigens are overwhelmed by the clonal population of T cells respondingto the dominant antigens.

In the case of cancer, chronic or latent infections, the local antigenprocessing/presenting and costimulatory environment impacts the primaryimmune response to the dominant antigens such that the T cell responseto the dominant antigens in the tumor or infectious agent is balancedtowards tolerance or ineffective response rather than a potent effectorresponse. Due to differences in antigen processing and costimulation(CD28 and cytokines), this could be accentuated in organs wheredendritic cells (DCs) are not the prevalent antigen presenting cells(APCs) unlike the body surfaces where DC are the predominant antigenpresenting cell. It is also well known that tumors and infectious agentscreate an immunosuppressive environment which is not optimal for astrong primary immune response. Alternatively, if a dominant antigenresults in cells which are reacting to self, tolerance is broken andautoimmunity ensues. Such tolerance could be broken by the presence of achronic or latent virus leading to a response (even chronically to weaksubdominant antigens). There are multiple associations of viruses withautoimmunity. The inflammation at the site leads to release of otherantigens while the viral antigens provide help to the T cells responsiveto the organ, causing autoimmunity. After the dominance hierarchy isestablished in the primary response and reinforced by memory, the immunesystem in the patient will effectively replicate the same response eachtime the antigen is present.

An ongoing immune response against a dominant epitope can diminish theresponse to a subdominant epitope (Wolpert E Z 1998, Kedl R M 2003). Thedominance/subdominance hierarchy can be somewhat fluid. For instance,deleting or silencing T cell responses against a dominant epitope canlead to the appearance of a previously undetectable response againstsubdominant epitopes (Van derMost R G et al. 1997, Andreansky S S et al.2005). Similarly, removal of a dominant sequence in an epitope does noteliminate the response to the antigen but rather results in the hostresponding more strongly to a previously subdominant epitope (Allan J Eand Doherty P C 1985, Mylin L M et al. 2000).

SUMMARY OF THE INVENTION

The present invention relates to a novel therapeutic for cancer, chronicinfections, autoimmune diseases, and transplantation based upon themodification of the immunodominance hierarch by therapeutic manipulationof cellular homeostasis.

Disclosed is a novel approach to rebalance the immune response toantigens to provide significant therapeutic benefit in, among others,cancer, chronic and latent infection, autoimmunity and transplantation.By generating immune responses to subdominant epitopes and subdominantantigens in a controlled microenvironment, the invention fundamentallychanges the nature of the immune response to the disease to one thatprovides therapeutic benefit. It can change the balance of the immuneresponse before or after a prior immune response has occurred to theantigen or even if there is one ongoing.

The present invention features a method comprising identifying adominant antigen or epitope and a subdominant antigen or epitope in apatient sample, cultivating a T cell capable of recognizing thesubdominant antigen or epitope, and treating a patient with an effectivenumber of the T cells to alter the immunodominance hierarchy of thepatient.

The present invention also features methods for altering theimmunodominance hierarchy of a patient comprising identifying at leastone subdominant antigen or epitope in a patient sample, cultivating a Tcell capable of recognizing the subdominant antigen or epitope, andtreating the patient with an effective number of those T cells toprovide therapeutic benefit.

In some aspects, the invention further comprises cultivating a T cell inthe absence of a dominant antigen or epitope. In other aspects, theinvention further comprises cultivating a T cell in the absence orpresence of agents that enrich suppressive T cells or responsive Tcells. Such agents can include, but are not limited to, growth factors,hormones, or other immune cells.

In some aspects, the invention further comprises administering theeffective number of T cells via intradermal administration.

In other aspects, the invention further comprises pre-treating thepatient with a conditioning agent to reduce the number of endogenous Tcells prior to treating the patient with the cultivated T cells. Theconditioning agent can be, but is not limited to, a chemotherapeuticagent.

In some aspects, the T cell is provided ex vivo from a patient.

The subdominant antigen or subdominant epitope is, for example, anantigen or epitope to which a cellular or humoral immune response is notdetectable or is only detectable at a low level. Alternatively, thesubdominant antigen or subdominant epitope is an antigen or epitope thatevokes a weaker tolerance or immune response than that of a dominantantigen or dominant epitope. The subdominant antigen is, for example, aviral antigen, a fungal antigen, a bacterial antigen, a parasiticantigen, a prion antigen, a tumor antigen, or an antigen associated withautoimmunity, allergy, inflammation, organ transplant rejection, orgraft versus host disease. The viral antigen is, for example, a chronicor latent viral antigen. The viral antigen is be from EBV, HPV, HSV,VZV, Hepatitis B, Hepatitis C, HIV, HTLV, CMV, RSV, or influenza. Thetumor antigen is, for example, a tumor-associated antigen, a tumorspecific antigen, or an antigen associated with cancer stem cells ormetastasis.

The present invention also features methods for identifying a dominantantigen or epitope and/or a subdominant antigen or epitope in a patientsample, cultivating a T cell capable of recognizing the subdominantantigen or epitope, wherein the T cell is a suppressive T cell, andtreating a patient with an effective number of said T cell to alter theimmunodominance hierarchy of the patient, thereby inducing tolerance inthe patient for treatment or prevention of an autoimmune disease,allergy, inflammation, organ transplantation rejection, or graft versushost disease.

The present invention also features methods for identifying a dominantantigen or epitope and/or a subdominant antigen or epitope in a patientsample, cultivating a T cell capable of recognizing the subdominantantigen or epitope, wherein the T cell is a responsive T cell, andtreating a patient with an effective number of said T cell to alter theimmunodominance hierarchy of the patient, thereby inducing a cytotoxicimmune response in the patient for treatment or prevention of aninfection or cancer. The infection is, for example, a bacterial, viral,parasitic, or prion infection.

In any of the methods of the present invention, treatment or preventionof a disease, infection, cancer, or medical condition includesalleviating or ameliorating at least one symptom of a disease,infection, cancer, or medical condition. Therapeutic benefit includesany alleviation, amerlioration, improvement, prevention, or treatment ofat least one symptom of a disease, infection, cancer, or medicalcondition.

In some aspects, the patient sample is a blood sample.

The present invention also features a method further comprisingre-profiling of the patient by assaying for a tolerance or humoral orcellular immune response in response to the subdominant antigen orepitope to determine if the therapy successfully rebalanced the immuneresponse.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are expressly incorporated byreference in their entirety. In cases of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples described herein are illustrative onlyand are not intended to be limiting.

Other features and advantages of the invention will be apparent from andencompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C show the results of a ⁵¹Cr release assay.

FIG. 2 shows the results of a ⁵¹Cr release assay.

FIGS. 3A and 3B show the results of a ⁵¹Cr release assay. FIG. 3C showsthe percentage of viable APCs.

FIG. 4 shows INFγ producing cells measured by Elispot.

FIGS. 5, 6A, 6B, 7 and 8 show the results of a ⁵¹Cr release assay.

FIG. 9 shows a mouse model of chronic hepatitis B.

FIG. 10 shows treatment of the mouse model.

FIG. 11 shows T cell responses to HBs and HBc.

FIGS. 12 and 13 show responses by method of administration.

FIG. 14 shows a hierarchy of antigens.

FIG. 15 shows responses by ICS.

FIG. 16 shows a hierarchy of antigens.

FIG. 17 shows immune response following an acute flair, then clearanceof hepatitis.

FIG. 18 shows a hierarchy of antigens following the clearance.

FIG. 19 shows that the T cells completely resolved the patient'sHepatocellular carcinoma (before treatment—left; 8 weekspost-therapy—right).

FIG. 20 shows antigens present in a patient's tumor.

FIG. 21 shows cells responding to the NY-ESO-1 antigen.

FIG. 22 shows antigens present after therapy in accordance with thepresent invention.

FIG. 23 shows the rebalancing of immunodominance hierarchy.

FIG. 24 is a CT scan from before and after T cell therapy in accordancewith the present invention.

FIG. 25 shows post treatment survival, progression free.

FIG. 26 shows a comparison of therapy by the present invention andRituxan+CHOP.

FIG. 27 shows a characterization of the T cells administered to theanimals.

FIG. 28 shows clinical disease scores for trial mice.

FIG. 29 shows the incidence of arthritis in trial mice.

FIGS. 30A, 30B and 30C show the histopathology of a normal rat, a ratimmunized with human proteoglycan and a rat treated with T cells.

FIG. 31 is a schematic of a bioreactor for use with the therapy of thepresent invention.

DETAILED DESCRIPTION A. Definitions

The term “antibody” is used in the broadest sense and specificallycovers human, non-human (e.g., murine) and humanized monoclonalantibodies (including full length monoclonal antibodies), polyclonalantibodies, multi-specific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they exhibit the desired biologicalactivity.

The term “antigen,” refers to a compound, composition, or substance thatcan stimulate the production of antibodies or a T cell response in ananimal, including compositions that are injected or absorbed into ananimal. An antigen reacts with the products of specific humoral orcellular immunity, including those induced by heterologous immunogens.The term “antigen” includes all related antigenic epitopes.

“Antigen presenting cells” or “APCs” are cells of the immune system usedfor presenting antigen to T cells. APCs include dendritic cells,monocytes, macrophages, marginal zone Kupffer cells, microglia,Langerhans cells, T cells, and B cells (see, e.g., Rodriguez-Pinto andMoreno (2005) Eur. J. Immunol. 35:1097-1105).

“Autoimmunity,” “autoimmune disease,” “autoimmune condition” or“autoimmune disorder” refers to a set of sustained organ-specific orsystemic clinical symptoms and signs associated with altered immunehomeostasis that is manifested by qualitative and/or quantitativedefects of expressed autoimmune repertoires. Autoimmune diseasepathology is manifested as a result of either structural or functionaldamage induced by the autoimmune response. Autoimmune diseases arecharacterized by humoral (e.g., antibody-mediated), cellular (e.g.,cytotoxic T lymphocyte-mediated), or a combination of both types ofimmune responses to epitopes on self-antigens. The immune system of theaffected individual activates inflammatory cascades aimed at cells andtissues presenting those specific self-antigens. The destruction of theantigen, tissue, cell type or organ attacked gives rise to the symptomsof the disease.

The term “cancer” refers to a disease or disorder that is characterizedby unregulated cell growth. Examples of cancer include, but are notlimited to, carcinoma, lymphoma, blastoma and sarcoma. Examples ofspecific cancers include, but are not limited to, lung cancer, coloncancer, breast cancer, testicular cancer, stomach cancer, pancreaticcancer, ovarian cancer, liver cancer, bladder cancer, colorectal cancer,and prostate cancer. Additional cancers known to those of skill in theart are also contemplated.

“Dominant antigen” or “dominant epitope” refers to an antigen or epitopethat evokes a strong tolerance or immune response, which may becharacterized by the presence of T cells specific for that antigen orepitope in an amount greater than about 70% of the total number ofresponding T cells.

The term “epitope” refers to a set of amino acid residues that isinvolved in recognition by a particular immunoglobulin, or in thecontext of T cells, those residues necessary for recognition by T cellreceptor proteins and/or Major Histocompatibility Complex (MHC)receptors. In an immune system setting, in vitro or in vivo, an epitopeis the collective features of a molecule, such as primary, secondary andtertiary peptide structure, and charge, that together form a siterecognized by an immunoglobulin, T cell receptor or HLA molecule.

“Hepatitis” refers to a medical condition defined by the inflammation ofthe liver.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, Calif. (1994).

An “immune response” refers to a response of a cell of the immunesystem, such as a B cell, T cell, or monocyte, to a stimulus. In oneembodiment, the response is specific for a particular antigen (an“antigen-specific response”). In one embodiment, an immune response is aT cell response, such as a CD4+ response or a CD8+ response. In anotherembodiment, the response is a B cell response, and results in theproduction of specific antibodies.

Immunodominance is the observation that in spite of a large number ofpossible epitopes (antigen fragments) in an antigen, the immune systemfocuses its response on a limited number of epitopes and can be orderedas a reproducible hierarchy (Sercarz et al. 1993). Immunodominance holdstrue for immune responses to artificial antigens, human virusesincluding influenza and vaccinia, and intracellular bacteria (Chen W S1994, Belze G T et al. 2000, Chen W 2000, Tscharke D C 2005). The finaloutcome of immunodominance is determined by a number of steps, includingMHC binding affinity, efficiency of cellular processing to generateappropriate MHC binding peptides, availability of TCRs to recognizecomplexes between the MHC binding peptides, and MHC followed by cellularimmunoregulatory mechanisms (Yewdell J W 2006, Sette A et al. 2009).

“Lymphocytes” refers to a type of white book cell that is involved inthe immune defenses of the body. There are two main types oflymphocytes: B cells and T cells.

“Major Histocompatibility Complex” or “MHC” is a generic designationmeant to encompass the histocompatibility antigen systems described indifferent species, including the human leukocyte antigens (“HLA”).

“Subdominant antigen” or “subdominant epitope” refers to an antigen orepitope that evokes a weaker tolerance or immune response than that of adominant antigen or dominant epitope.

The term “treatment” refers to a clinical intervention made in responseto a disease, disorder or physiological condition manifested by apatient or to be prevented in a patient. The aim of treatment includesthe alleviation and/or prevention of symptoms, as well as slowing,stopping or reversing the progression of a disease, disorder, orcondition. “Treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already affected by a disease or disorder or undesiredphysiological condition as well as those in which the disease ordisorder or undesired physiological condition is to be prevented.

“Tumor” refers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues.

“______-mer” refers to a linear sequence of ______ amino acids thatoccur in a target antigen.

B. Assays that Recognize/Distinguish the Subdominant Epitope from theDominant One

The patient's tumor or infection is first assayed for the presence of apanel of tumor associated, viral or other antigens. This is generallydone by immunohistochemistry on the tumor biopsy or FACS in the case ofhematologic malignancies. The patient's tumor or infection is assayedfor the presence of a panel of tumor associated, viral or otherantigens. This is generally done by immunohistochemistry on the tumorbiopsy or FACS in the case of hematologic malignancies. The patient'sblood is drawn and tested for both humoral and cellular immune responseto the antigens which are present. The antigens to which the immuneresponse is not detectable or detectable at a low level are proactivelyselected to grow T cells in vitro. After these T cells are grown, theycan be tested for response to the antigens re-infused, and the patient'sblood can be assayed for the response to the antigens. In this way, thepatient's immune response can be effectively rebalanced to providetherapeutic benefit. In a preferred embodiment, the assays of humoralimmunity can include but is not limited to an ELISA assay. In apreferred embodiment, the assay of cellular immunity can include but isnot limited to Intracellular cytokine staining for cytokines (ICS),including but not limited to Interferon γ (INFγ) and Tumor necrosisFactor α (TNFα). The subsets of T cells responding (e.g., CD8, CD4,Treg) can also be assayed in this assay. Alternatively, the assay ofcellular immunity can include but is not limited to ELISPOT assay forINFγ or TNFα. In an alternative embodiment, the Elispot or ICS can assayfor IL-4, IL-12 (TH2 and TH1), IL-10 (Treg) or IL-21 (T follicularhelper cell subset). In still another embodiment, the cellular immuneprofiling assay can be Intracellular Staining (ICS) for these or othercytokines. The antigens tested can be full antigens, antigens withepitopes deleted or dominant or subdominant epitopes. In the case ofepitopes, bioinformatics software can be used to predict epitopes whichwould bind to the patients MHC and these epitopes are then assayed. Inone embodiment, this software is the Net MHCpan or the consensus epitopeimmunoinformatics software described in the assay section. In the caseof epitopes, tetramer binding can be used as an alternative assay toquantify the CTL providing one knows the HLA type. Tetramers includingthe peptides are combined with the cells and cells stained and FACS usedto determine the % of cells recognizing each tetramer. This is useful ifthe patient is of a known HLA type such as HLA A2. However, thepreferred method is the ELISA for humoral response and ICS or Elispotfor the cellular response. In a preferred embodiment, the CTL's aregenerated from peripheral blood. Alternatively, the CTL's are generatedfrom the tumor infiltrating lymphocytes (TIL) or from the DTHsurrounding the injection site.

DTH infiltrating lymphocytes can be prepared by taking a 4 mm punchbiopsy from the skin minced in RPMI medium 1640 with 10% FCS (CSL).Single cell suspensions were stimulated with 1 μ/ml phytohemagglutinin(Sigma) and cocultured with irradiated autologous PBMC'S with 10 IU/mlIL-2 (Cetus) and 10 ng/ml IL-7 (Peprotech, Rocky Hill, N.J.). Medium wasreplenished each 2-3 days.

For humoral immune response profiling, serum from the patient isserially diluted 1:4 from 1/100 to 1/100000 and used in a standard ELISAwith purified recombinant tumor antigens (generally made in E. coli).From 2 to 10000+ antigens can be assayed. One microgram of each purifiedprotein is absorbed to microwell plates (Nunc) overnight at 4 degrees C.Plates are washed with PBS and blocked with 2% FCS/PBS. Patient serum isdiluted in 2% FCS/PBS and added for 2 hours. Plates are washed and goatanti-human IgG-AP (Southern Biotechnology Assoc) is added. Plates arewashed, incubated with Attophose substrate (JBL Bioscientific) for 25min, and immediately read (CytoFluor 2350, Millipore). Readout is UVAbsorbance.

There are two methods for cellular response profiling. The first methodinvolves the Enzyme-linked immunosorbent spot (“ELISPOT”) assay forINFγ. Ninety-six well polyvinylidene diflouride backed plates(Millipore, Bedford, Mass.) are coated with 5-15 μg/ml of anti-INFγmonoclonal antibody 1-DIK (MABTECH, Stockholm, Sweden) at 4 degrees C.overnight. The wells are washed and blocked with 5% human AB serum(Valeant Pharm). 5×10⁶ PBMCs (or 5×10⁵ CTL's when the assay is performedpost in vitro expansion) are added per well with peptide mixes 2 μM eachfrom each of the antigens. Incubate overnight (18 hours) at 37 degreesC. 5% CO₂. Cells are discarded and the wells are washed with PBScontaining 0.05% Tween 20. 1 μg/ml biotinylated anti-INFγ monoclonalantibody 7-B6-1 (MABTECH) is incubated for 2-4 hours at room temperaturefollowed by streptavidin conjugated alkaline phosphatase (MABTECH orSigma Aldrich) for 2 more hours. This is followed by a 30 minutereaction with 5-bromo-4-chloro-3-indolylphosphate and nitro bluetetrazolium from the alk-phos substrate kit (Bio-Rad Richmond, Calif.).The spots are to be counted using a dissection microscope (SZ CTVOlympus microscope). Spots can also be counted on an AIDELISPOT reader(Autoimmun Diagnostika, Strassberg, Germany). Each spot is a cellreported as spot forming cells (SFC)/10⁵ PBMC's. 10 μg/ml PHA can beused as a positive control; cells alone without peptide can serve as thenegative control.

The second method involves intracellular cytokine staining for INFγ andTNFα. 5×10⁶ PBMC's (or 5×10⁵ CTL's when the assay is performed post invitro expansion) are plated in 100 μl PBS 1% FCS 96 well plates togetherwith the peptides (10⁻⁵ to 10⁻⁹ M final concentration) for each of theepitopes or antigens being studied. After 6 hours of incubation in thepresence of IL-2 (150 U/Ml), 50 μM β mercaptoethanol and brefeldin A (1μg/ml) or Golgi Plug (BD Biosciences, San Diego, Calif.) (both of thelatter components to increase accumulation of INFγ or TNFα in respondingcells), cells are pelleted, washed in 200 ml PBS 1% FCS and then labeledwith stain for surface antigens (CD4 fluorescein isothiocyanate and CD8allophycocyanin 0.25 μ/ml (Pharmingen, Becton Dickinson) for 30 minutesat 4 degrees C. (for 30 min on ice). After a wash, cells arepermeabilized with Cytofix/Cytoperm for 20 min on ice and then stainedwith a phycoerythrin conjugated anti-INFγ(0.4 μg/ml) or anti-TNFα (0.8μg/ml) antibody (Pharmingen, Becton Dickinson). The cells are thenwashed, fixed and resuspended in PBS 1% FCS and tested on a FACScan flowcytometer and analyzed using Cell Quest software. Alternatively, FACSCanto (Becton Dickinson) can be used. Other cytokines, including but notlimited to IL-12 and IL-4, can be assayed to measure TH1 or TH2 subsets.Cytokine panel to give a broader assay of T cells could measure IL-12,IFNγ, IL-4, IL-10 and IL-17. T follicular helper cells can be measuredas CD4⁺, CXCR5⁺, ICOS⁺ cells. B cells can be measured as CD19⁺ and B220⁺cells. IL-21 in the T cells should be associated with B cell activationand affinity maturation of antibodies so this could be used to studythis as well. As an alternative for profiling INFγ, IL-4 (BDBiosciences) IL-12, IL-10, IL-17 (R&D Systems), and IL-21 antibody (R&Dsystems), ELISPOT can also be used. ICS actually profiles the % of CD8or CD4 T cells responding to different antigens or epitopes. Other cellsubsets can be analyzed as well including Treg. A cytochrome labeledCD25 monoclonal antibody can be used as a surface marker of most Tregs.Alternatively, cytochrome labeled Human FoxP3 monoclonal antibody clone259D/C7 from BD Biosciences is used to stain the cells postpermeabilization to measure % Treg cells and their status. IL-10 canalso be assayed.

NetMHCpan is a bioinformatics method for quantitative predictions ofpeptide binding to HLA-A and -B (Nielsen M 2007). A consensus epitopeprediction approach has also been developed (Mouaftsi M 2006). Thesemethods can be used to sort all of the potential MHC I epitopes for anantigen and rank the top 1% of peptides and thus predict epitopes. Thesepredicted epitopes would then be synthesized as 9-10 mer peptides andtested (e.g., to patient PBMCs or in a transgenic mouse for the HLA typeof interest).

Tetramers with specific MHC (e.g., HLA A A2) are synthesized togetherwith 8 mer peptide epitopes (in the case of class I MHC and 15 merpeptide epitopes in the case of MHC Class II. The cultured T cells arestained with the tetramer diluted 1/200 at room temperature for 20 min;anti CD8 antibody was then added and stained for a further 30 minutes.Cells were then washed and 100,000 acquired on FACS Calibur (BDBiosciences) and analyzed with Flowjo software (Tree Star).

Following therapy, the patient is re-profiled by assaying for atolerance or humoral or cellular immune response in response to thesubdominant antigen or epitope to determine if the therapy successfullyrebalanced the immune response.

C. Rebalancing the Immune Response

In one embodiment of the present invention, the immune response can berebalanced by growing T cells from a patient ex vivo to a subdominantantigen or subdominant epitopes on an antigen followed by infusion oradministration of these T cells into the patient. T cells to subdominantantigens or epitopes are grown in tissue culture, ex vivo (away from thepatient's immunoregulatory milieu). After growing enough cells tooverwhelm the previously dominant cells, the cells are re-infused intothe patient to skew the cellular balance and therapeutically switch thedominance hierarchy. In a further preferred embodiment, this number of Tcells introduced de novo as therapy is greater than 5% of the T cellsresponding to the antigen, infectious agent, tumor or organ. The ratiocan be further skewed to favor the infused cells by pretreatment of thepatient with conditioning agents which reduce the number of endogenous Tcells (i.e., chemotherapy).

The present invention involves methods to optimize the growth of T cellsto subdominant antigens in tissue culture. In one embodiment, the cellsare grown in the absence of dominant antigens. This is accomplished byselection of a professional antigen presenting cell which has not beenexposed to dominant antigen and the modification of the antigens toeliminate dominant epitopes or other components which limit the abilityof an antigen to be processed.

As the therapeutic method requires T cells to be enriched for andideally fully responsive to subdominant antigens/epitopes, efficientmethods for the growth of such T cells are important. The growth ofother T cells to dominant epitopes and antigens increases the time inculture required to generate enough specific cells to skew this ratio.Moreover, the cells growing out to dominant epitopes is working againstthe achievement of the proper ratio upon reinfusion. Therefore, T cellculture methods which specifically limit the introduction of dominantantigens or epitopes have been developed. For example, while the methodis broadly applicable to all tumors, it has distinct advantages for EBVmalignancies because it does not use EBV transformed B cells (whichexpress the EBV dominant antigens/epitopes). It is also more reliablewhen administered to cells in which a significant % of the CTL's areresponding to subdominant antigens/epitopes.

Monocyte-derived dendritic cells are generated in vitro from peripheralblood mononuclear cells (PBMCs) from a patient. In a preferredembodiment, plating of PBMCs for 2 hours in a tissue culture flaskpermits adherence of monocytes. In an alternative embodiment, CD14⁺magnetic beads can be used to isolate dendritic cells from PBMC's(Miltenyi Biotec, Auburn, Calif.). At this point the nonadherent cellsare removed and frozen at −80 to later serve as a source of T cells.Treatment of the adherent monocytes with interleukin 4 (IL-4) andgranulocyte-macrophage colony stimulating factor (GM-CSF) leads todifferentiation to immature dendritic cells (iDCs) in about a week.Subsequent treatment with tumor necrosis factor (TNF) or macrophageconditioned media for 2 days further differentiates the iDCs into maturedendritic cells. These cells are then pulsed with a peptide or a plasmidcontaining a subdominant antigen for 2 hours and then the PBMC's arethawed and added to the pulsed dendritic cells. After a few hours, thecells are pooled and resuspended in media containing IL-2 or IL-15 (withIL-15 being preferred) to generate in vitro expansion of the T cellswhich have recognized the antigen. For certain protocols, IL-7 and IL-15are added to increase T cell survival. For other protocols, cultureconditions are adjusted to optimize growth of certain subsets of Tcells. For example, IL-12 can be added to polarize to TH1 cells.Alternatively, IL-4 can be added to polarize to TH2. In certainprotocols, IL-6 can be added to prevent the growth of Treg. In stillanother variation which is useful in autoimmune or organ transplantationapplications, low level IL-2⁺ rapamycin can be added to accentuate thegrowth of Treg. More detailed protocols are outlined in the variousexamples and in vivo comparison of the cells produced with protocols topolarize the cells to certain T cell subsets is described in Example 3,FIG. 3. If the T cells are grown in a tissue culture flask, media mustbe replaced at day 14 and day 21. However, in a preferred embodiment, abioreactor can be used to mitigate this need, e.g., a gas permeablebioreactor such as Grex (Wilson Wolf) or Hyperstack (Corning).Generally, enough cells to be administered to a patient can be generatedwithin 2 to 6 weeks as opposed to 12-24 weeks with traditional methods.

In another embodiment of the invention, the inventors have developed Tcells for adoptive transfer against antigens which have been deleted fortheir dominant epitopes and demonstrate that more T cells are generatedto subdominant epitopes. Such proteins or corresponding DNA vaccines canbe used to generate T cells with a broad immune response againstsubdominant epitopes. This approach should be broadly applicable acrossa wide range of diseases to achieve a balance of the immune responsetowards subdominant epitopes including but not limited to EBV, cancer,HIV or hepatitis. In another embodiment, the antigen, or aplasmid/recombinant vaccine encoding it, is used to vaccinate thepatient to induce a de novo broad immune response to the subdominantantigen. In another embodiment, the subdominant reactive T cells can beadministered followed by a vaccination with the subdominant antigen toboost the response. In still another embodiment, the approach could beused therapeutically or prophylactically wherein a patient's immuneprofile can be determined to measure risk of coming down with diseasesand the patient can then be primed to appropriate subdominant antigensusing any of the approaches disclosed herein.

In an alternative embodiment, the grown T cells induce tolerance toprevent or treat an autoimmune disease, allergy, inflammation, organtransplantation rejection, or graft versus host disease. Depending uponthe type of T cells desired, culture conditions can be modified topreferentially grow, or enrich for, the relevant subset including butnot limited to CD8, CD4, TH1, TH2, or Treg. For example, T cells can begrown in presence or absence of certain growth factors, cytokines,drugs, small molecules, or other immune cells. In a preferredembodiment, subdominant antigen reactive T cells are generated fromPeripheral Blood Mononuclear Cells (PBMC's) in tissue culture in thepresence of a stimulated professional Antigen Presenting Cell (e.g.,monocyte derived dendritic cell, macrophage or EBV immortalized B cell).

In another embodiment, various techniques are used to modify antigenprocessing to favor subdominant epitopes. In one embodiment of theinvention, this is accomplished by modification of the antigens toeliminate dominant epitopes, regions which inhibit antigen processing,or to limit the number of dominant or subdominant epitopes presented atone time to an antigen presenting cell. These modifications increase theresponse and diversity of subdominant epitopes recognized (Example 1,FIG. 5). In an alternative embodiment, the modified LMP1, LMP 2 andEBNA-1 sequences can be delivered to the APC using a viral vector suchas adenovirus or vaccinia virus. In the case of other antigens (LMP1 andEBNA-1), eliminating regions of the protein which lead to poor antigenprocessing greatly enhances the immune response to subdominant epitopeson those antigens (Example 1, FIG. 6). The modified LMP1, LMP2 andEBNA-1 sequences can be delivered to the APC using peptides, proteins,plasmids or viral vectors such as adenovirus or vaccinia.

In an alternative embodiment, a proteosome antagonist may be added tothe APC's and antigen during CTL production to increase the number ofsubdominant epitopes recognized and enhance the response to thesubdominant antigen (Example 2, FIG. 7). There are many availableproteosome antagonists having different mechanisms (e.g., bortezomib,clioquinol, lactacystin, epoxomycin, MG-132, MLN9708, carfilzomib(PR-171)).

In an alternative embodiment, an antigen may be administered incomplexes with antibodies having various isotypes regarding responses tosubdominant determinants (Example 3, FIG. 13). The antigen is injectedwith an antibody binding a determinant flanking the intended T cellepitope to target professional antigen presenting cells and directantigen processing to the flanking epitopes.

In another embodiment of the present invention, plasmids containingsubdominant epitopes or antigens are used to generate T cells which areadministered or are directly administered into the patient eitherdirectly by various routes of administration, in combination with INFγ,IL-21 or other cytokines or on pulsed dendritic cells to induce aresponse to subdominant antigens. IFN-gamma or other cytokines may beinduced before T cell stimulation to increase T cell responsiveness tosubdominant epitopes and modify the immunodominance hierarchy.

In another embodiment, the route of administration is modified to alterthe immunodominance hierarchy. The route of administration of a vacciniaresponse determines the degree of dominance of the dominant determinant.It has been found that when administered intraperitoneally, the dominantdeterminant accounted for only a quarter of the response as opposed tohalf, as was the case with intradermal administration (Tscharke D C etal. 2006, Tscharke D C et al 2005). As shown in Example 3, FIG. 11,administration of antigen by the IM route develops a stronger responseand broader response to the subdominant epitope than does IP or IVroutes. Thus, modifying the route of administration is another in vivomechanism which the inventors claim to modify the immunodominancehierarchy. In a preferred embodiment, the T cells cultivated aredelivered by intradermal administration. By targeting different APCs(e.g., macrophages, dendritic cells), the route of administrationchanges the dominance hierarchy (Example 3, FIG. 12).

In a preferred embodiment, the antigens are viral antigens particularlylatent viral antigens or chronic viral antigens with subdominantepitopes on them. For example, the viral antigens are from a virusselected from the group comprising: EBV, HSV, VZV, Hepatitis B and C,HIV, and HTLV. The viral antigens are, for example, EBV LMP1, LMP2,EBNA-1, HPV E6, or HPV E7. For example, the viral antigens areassociated with EBV, HSV, VZV, Hepatitis B and C, HIV, and HTLV, CMV,RSV, or influenza. In another embodiment, the antigens are antigens onother chronic and latent infectious agents, for example, agentsassociated with, bacteria, fungi, parasites, or prions. In still anotherembodiment, the antigens are tumor antigens including but not limitedto: tumor associated antigens, tumor specific antigens, antigensassociated with cancer stem cells or metastasis. In other embodiments,the antigens are associated with autoimmunity, allergy, inflammation ororgan transplantation rejection or graft vs. host disease.

In one embodiment, the immunodominance hierarchy of a patient is alteredby identifying a dominant antigen or epitope and a subdominant antigenor epitope in a patient sample, cultivating a T cell capable ofrecognizing the subdominant antigen or epitope, and treating a patientwith an effective number of the T cells.

In one embodiment, the immunodominance hierarchy of a patient is alteredby identifying at least one subdominant antigen or epitope in a patientsample, cultivating a T cell capable of recognizing the subdominantantigen or epitope, and treating the patient with an effective number ofthose T cells to provide therapeutic benefit.

In another embodiment, the T cell is a responsive T cell, and treating apatient with an effective number of said T cell to alter theimmunodominance hierarchy of the patient, thereby inducing a cytotoxicimmune response in the patient for treatment or prevention of aninfection or cancer. The infection is, for example, a bacterial, viral,parasitic, or prion infection.

In any of the methods of the present invention, treatment or preventionof a disease, infection, cancer, or medical condition includesalleviating or ameliorating at least one symptom of the disease,infection, cancer, or medical condition.

D. Therapeutic Methods

1. Cancer

Work Flow of Clinical Use of Immune Profiling of Dominant andSubdominant Antigens

Step 1: Tumor Biopsy (Immunohistochemistry) or Blood (IHC, FACS orElisa) Antigen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10

Result: Antigens 1, 2 & 6 on tumor, others in panel not

Step 2: Immune Response Profiling

Humoral Profile Cellular Profile ELISA on Serum Elispot or ICS (for IFNgat least, also IL-10, IL-4 and IL-12, IL-21 to assay T cell subsets) onPBMCs stimulated with each antigen

Result: Antigen 1 strong response (dominant); Antigen 2& 6 No/ModestResponse (subdominant)

Step 3: Grow T Cells (CD8 and CD4) to Subdominant antigens in vitro

Result: T cells responsive to antigens 2& 6

Step 4 (optional once therapy is well established): Confirm >5% T cellsgrown respond to subdominant antigens using Cellular Immune Profiling(Elispot or ICS)

Result: 25% of the T cells respond to Antigens 2 & 6

Providing at least 5% of T cells respond to subdominant antigens,proceed to step 5

Step 5: IV Infuse cells into patient with or without prior conditioning(e.g., cyclophosphamide)

Step 6 (may become optional once therapy is well established): IsolatePBMC's from Blood 2-3 weeks post infusion and Profile Immune Response

Result: Cellular Profile Antigen 1 No/Moderate response; Antigens 2 &Strong response (dominant) to at least 1 of the antigens

Step 7: Assess Clinical responses

RECIST (CR, PR); Survival or Progression Free Survival

Result: Improved response rate and survival

In Example 4, this systematic method is used to treat melanoma. InExample 5, the method is applied to treat lymphoma with the multiplasmidLMP2. In fact the T cell therapy changes the natural course of lymphomafrom one that is relapsing remitting to one that is a durable remission.This antigen as well as deleted LMP1 and EBNA-1 can also be used totreat other tumors that include EBV antigens including Nasopharyngealcarcinoma, Burkitts Lymphoma, CLL, Hodgkins, and some gastric canceramong others. In Example 3, a similar method is used to treatHepatocellular carcinoma. These examples are incorporated into theinvention by reference and demonstrate that rebalancing of the immuneresponse towards subdominant antigens is broadly applicable to all formsof cancer.

2. General Use of a Large Number of Subdominant Antigens on a Tumor toProduce a Pan Tumor Type Therapy

In another embodiment, the inventors propose that if enough subdominantantigens can be identified for a particular type of tumor, that thattype of tumor could be treated with T cells to multiple subdominantantigens without the need to test the patient. Similarly, targetingmultiple antigens on the same tumor would decrease resistance, e.g.,combination chemotherapy. For example, the ability to develop a single Tcell line targeting the subdominant antigens EBV LMP2 which is on 40% oflymphomas and surviving (which is on 50% of lymphomas) would allow oneto target approximately 80% of lymphomas with a T cell line specific tothese 2 antigens. Clinical testing of the Pan-lymphoma product isdemonstrated in Example 5. By year 3, progression free survival ofpatients treated with the Pan lymphoma product is comparable to theproduct where antigens were tested before therapy. This could bebecause, like combination chemotherapy, the response of CTL's tomultiple antigens on the same tumor could decrease the likelihood ofescape. The ability to treat all lymphoma (not having to only treat thatsubset which is positive for a single antigen) with a single T cellproduct is a novel product concept. In other embodiments of theinvention, the inventors also claim similar Pan-cancer products forvirtually any cancer.

3. Chronic Infections

Hepatitis and Hepatocellular Carcinoma

In patients with chronic Hepatitis, HBs antibodies are not generated butHBc antibodies are (Ganem D et al 2004). In patients with acutehepatitis, antibodies to both antigens are generated. >90% of neonatesand 30% of children ages 1-5 develop the chronic form while adultsacutely clear the virus >90% of the time. 95% of Hepatocellularcarcinoma is associated with chronic infection with Hepatitis B virusand. HBsAg often is on the cell surface of HCC. Given theseobservations, the inventors chose to study whether HBs was subdominantin hepatitis and hepatocellular carcinoma patients and if the growth ofT cells could generate CTLs to HBs antigen which could rebalance theimmune response to subdominant antigens and be therapeutic.

Based upon this finding, we administered the CTL's grown to HBs Ag totreat chronic hepatitis in an animal model of hepatitis and ultimatelyin HBV associated HCC patients. In the animal model, HBVtgRAG cells wereadministered and the tested. In the animal which received control cells,chronic Hepatitis developed. Furthermore, when the animal developedhepatitis and received CTL's to subdominant antigens, the animaldeveloped acute hepatitis but cleared the hepatitis virus (Example 3,FIGS. 9-11).

Given the encouraging animal data, patients with hepatitis were treatedwith the T cell rebalancing therapy.

Work Flow of Clinical Use of Immune Profiling of Dominant andSubdominant Antigens

Step 1: Immune Response Profiling Hepatitis Surface Antigen andHepatitis Core Antigen

Humoral Profile Cellular Profile ELISA on Serum Elispot or ICS (for IFNγat least, also IL-10, IL-4 and IL-12, IL-21 to assay T cell subsets) onPBMCs stimulated with antigen

Result: Hbc strong response (dominant); Hbs No/Modest Response(subdominant)

Step 2: Grow T cells (CD8 and CD4) to Subdominant antigen in vitro

Result: T cells responsive to HBs antigen

Step 3 (optional once therapy is well established): Confirm >5% T cellsgrown respond to subdominant antigen using Cellular Immune Profiling(Elispot or ICS)

Result: 25% of the T cells respond to HBs Antigen

Providing at least 5% of T cells respond to subdominant antigens,proceed to step 4

Step 4: IV Infuse cells into patient with or without prior conditioning(e.g., cyclophosphamide) Step 5 (may become optional once therapy iswell established): Isolate PBMC's from Blood 2-3 weeks post infusion andProfile Immune Response

Result: Cellular Profile HBc No/Moderate response; HBs Strong response(dominant)

Step 6: Assess Clinical responses

Result: Clearance of Infection

Immune profiling was conducted on 5 patients with HBV and HCC. Asexpected, ELISA demonstrated high titers of antibody to hepatitis B coreantigen (HBc Ag) but not to hepatitis B surface antigen (HBs Ag)(Example 3, FIG. 17). However, when PBMC's were induced into dendriticcells, pulsed with hepatitis B core antigen (HBc Ag) and hepatitis Bsurface antigen (HBs Ag) and used to grow CTL's from PBMC's from thesame patient, a surprising result was observed. By frequency of IFNproducing T cells on Elispot a hierarchy was observed: no-antigen (10sfc)<HBsAg(15 sfc)<HBcAg(45 sfc) (Example 3, FIG. 14). This indicatesthat Hepatitis B surface antigen was indeed subdominant relative toHepatitis core. Furthermore, it appeared that one epitope (FLL) of HBswas dominant by Elispot (Example 3, FIG. 14) and by ICS (Example 3, FIG.15). The peptide containing this epitope was excluded from the pepmixthat was used to grow cells from that patient.

In patients with HCC, 3/5 patients who received the T cells tosubdominant HBs (Example 3, FIG. 16) cleared the HBs antigen anddemonstrated CR's to the HCC following a transient elevation of Alaninetransaminase (ALT) in Liver Function Tests (Example 3, FIGS. 17 & 19).When PBMC's from those patients were tested 2 weeks post administration,the dominant response was to HBs rather than to HBc and to thepreviously subdominant epitopes of HBs (Example 3, FIG. 18) indicatingthat the patients had rebalanced their immunodominance hierarchy. As aresult of the therapy and the switch in immunodominance hierarchy wasproviding the therapeutic benefit (Example 3, FIGS. 17 & 19).

Thus, T cell adoptive immunotherapy to subdominant antigens can be usedto eradicate chronic viruses and to treat cancers on which they arepresent. Further, immune profiling to direct the production of T cellsto subdominant antigens and epitope opens a novel therapeutic avenue inmultiple diseases.

Virtually any viral, bacterial, fungal, prion, parasitic or otherinfectious disease can be treated including but are not limited toHepatitis C virus and HIV virus. Other chronic infectious disease canalso be treated using rebalancing. For example, Mycobacterialtuberculosis is a chronic infection in many patients but onlyreactivates when the immune system is repressed. In its dormant state,it is contained by a granuloma usually in the lung. Recent studies ofthe proteome of the mycobacterial phagosome indicate that MTB repressesantigen presentation in dendritic cells to a greater degree thanmacrophages (Li et al. 2011). To this end the inventors proposed thatrebalancing the immune system to respond to subdominant epitopes is abroad therapeutic approach for virtually any cancer or infectiousdisease.

4. Autoimmunity

Epidemiological studies demonstrate that the risk of patients developingmultiple sclerosis correlates with EBV antibody titers. By followinghundreds of thousands of individuals before they were infected with EBVand following up with them for several years post initial infection,Ascherio et al. were able to study the 305 patients who developed MS.Their risk increased sharply following EBV infection (Ascherio A et al.2010). Memory CTL's responsive to EBNA-1 400-641 were elevated in MSpatients relative to other EBV antigens when compared to healthyindividuals who were also EBV carriers (Lunemann J D et al. 2006).EBNA-1 specific Th1 cells appear capable of sustaining autoimmunity bycross recognition of auto-antigens or bystander mechanisms. Further,transgenic mouse studies suggest that B cells expressing LMP2a bypassnormal tolerance checkpoints and enhances development of autoimmunedisease (Swanson-Mungerson M 2007). As such, while we have studied Tcells reactive to EBV subdominant epitopes as a treatment for EBV andEBV related cancers, T cells reactive to EBV subdominant epitopes mayalso be useful in reinstating balance in autoimmunity. By growing andintroducing the T cells responsive to EBV latent antigens (EBNA-1, LMP1and LMP2), the inventors propose that the immune response can berebalanced, reinducing tolerance in the autoimmune diseases with an EBVassociation. The same would hold true for other viruses associated withother autoimmune disease. For example, Picornaviruses such asCoxsackievirus B3 cause myocarditis/dilated cardiomyopathy, type 1diabetes, encephalitis, myositis, orchitis, hepatitis.

In still other embodiments, T cells can be generated against antigensassociated with organs in which organ specific autoimmunity isdeveloped. Because the phenomenon of immunodominance involves epitopesfrom all of the antigens which are being processed at any one time,there is not a completely required need to know the exact autoantigenwhich is driving the autoimmune response at that time. For example,generating and introducing T cells to reactive subdominant epitopes ofcollagen into the inflamed joint of rheumatoid arthritis patients, theongoing immune response will be rebalanced and tolerance will bereinstated. It is contrarian and unexpected to think that introducingactive T cells into an inflammatory site would be beneficial, butaccording to our operative model, the rebalancing of the immune responsein that site will reinstate appropriate control over the aberrant immuneresponse. The treatment of autoimmune diseases is another embodiment ofthe invention.

5. Treg In Vitro Expansion

One of the principles of immune regulation is the balance of Tregs toother subsets of T cells. Another embodiment of the invention ispolarizing the ex vivo T cell growth towards Treg cells. In this case,instead of targeting subdominant epitopes, Treg to dominant epitopes aregenerated as another way to rebalance the immune response. In oneembodiment, the Treg T cells so generated are used as the therapeuticproduct alone. In another embodiment, the Treg subset is used incombination with T cells from other subsets grown to subdominantepitopes or subdominant antigens While Tregs have been expanded in a nonantigen specific way using FACS sorting followed by expansion withanti-CD23 and anti-CD28 coated beads (Putnam et al. 2009), to date noone has expanded Tregs in an antigen specific way for adoptiveimmunotherapy. Described herein are methods which are useful in theestablishment of Tregs specific for different antigens especially thosewhich are dominant in the immune response observed in autoimmunedisease, graft vs. host disease or transplant rejection.

Tregs were isolated from PBMC's of autoimmune or Transplant patients byFACS sorting (BD FACS Aria II high speed cell sorter) using an aseptictechnique in a GMP clean room using CD4-PerCP(SK3), CD127-PE(hIL-7R-M21), CD25 APC (2A3), CD45RA-PE.Cy7 (L48) and CD45RO-PE.Cy5(UCHLI). CD4⁺ CD127^(lo/)-CD25⁺ and CD4⁺ CD127^(lo)/− T cells weresorted and collected in 3 ml X-Vivo 15 media (Lonza, Walkersville, Md.)containing 10% human heat-inactivated pooled AB serum (ValleyBiomedical, Winchester, Va.). (Alternatively, Tregs can be separatedusing magnetic beads coated with the same antibodies (Miltenyi Biotec,Auburn, Calif.). These cell were plated at 2.5×10⁵ Tregs per well in a24 well plate (Costar, Cambridge, Mass.), each well containing dendriticcells prepared from the PBMC's (as described for T cell stimulationabove) which had been pre-pulsed with dominant antigen at a Treg:APCratio of 1:5. Following 18 hours incubation, cells received rapamycin(100 ng/ml; Wyeth, Madison, N.J.), from day 1 to 7 in culture. At day 2,culture volume was doubled and 300 units/ml IL-2 was added (Chiron,Emeryville, Calif.). Cells were resuspended and fresh media and IL-2were added days 2, 5, 7, 9 and 12. On day 9 cells were restimulated withpeptide pulsed dendritic cells. Alternatively, anti-CD23/anti-CD28coated microbeads (Invitrogen, Carlsbad, Calif.) can be used for thissecond stimulation. Further, the gas permeable bioreactor (such as Grex)can be used to perform this cell growth with fewer manipulations in aclosed system and improve the kinetics of growth. In another variationIL-10 is added at the time of stimulation with the coated beads to drivefurther differentiation of the Treg to Treg1 which secrete high levelsof IL-10 and regulate TH1 and Th2 responses.

Work Flow of Clinical Use of Immune Profiling of Dominant andSubdominant Antigens in Autoimmunity

Step 1: Immune Response Profiling

Humoral Profile Cellular Profile ELISA on Serum Elispot or ICS (for IFNγat least, also IL-10, IL-4 and IL-12, IL-21 to assay T cell subsets) onPBMCs stimulated with each antigen

Result: Antigen 1 strong response (dominant); Antigen 2 No/ModestResponse (subdominant)

Step 2: Grow T cells (CD8 and CD4) to Subdominant antigens in vitro;Grow Treg to dominant antigens in vitro

Result: T cells responsive to subdominant antigens; Treg responsive todominant antigens

Step 3 (optional once therapy is well established): Confirm >5% T cellsgrown respond to subdominant antigens and Treg grow to dominant antigensusing Cellular Immune Profiling (Elispot or ICS)

Result: 25% of the T cells respond to Antigens

Providing at least 5% of T cells respond to subdominant antigens and/orat least 5% of Treg cells respond to dominant antigen, proceed to step 4

Step 4: IV Infuse cells into patient with or without prior conditioning(e.g., cyclophosphamide)

Step 5 (may become optional once therapy is well established): IsolatePBMC's from Blood 2-3 weeks post infusion and Profile Immune Response

Result: Cellular Profile Antigen 1 No/Moderate effector response;Antigens 2 & Strong Treg response (dominant)

Step 6: Assess Clinical responses

In autoimmunity (MS—flare ups decrease, Rheumatoid Arthritis—jointswelling reduced, Asthma—number of attacks decreases, Early Type Idiabetes, pancreas is maintained), In transplant-organ rejection rateand Graft vs. host disease decrease

Result: Improved clinical outcome

6. Administration of Tregs to Dominant Antigens in Autoimmunity orTransplant

In still another embodiment, Treg against dominant antigens could becombined with T cell of the TH1, TH2 or CTL subsets which are themselvesresponsive to subdominant antigens. Such a combination would more fullyswitch the balance of the response towards subdominant epitopes. Theinventors have demonstrated synergy between the two types of T cells inarthritis (Example 6).

7. Transplant

In an alternative embodiment, Treg are grown using the above Tregculture conditions from PBMC's of healthy donors using dendritic cells(or irradiated PBMC's) from other healthy donors. In this wayalloreactive Treg lines to each of the MHC are established and banked.As we discussed above, 80% of the MHC could be covered with Treg linesgenerated against 20 to 50 MHC haplotypes. Each of these lines could befrozen in single dose aliquots at −80 degrees C. When an organ or BMT isperformed, 5×10⁷ cells/m² Treg reactive to the mismatched MHC are alsotransplanted into the patient. In this way, the allogeneic rejection orgraft vs. host disease is mitigated.

8. Automated Immune Profiling Assays and Closed System Cell CultureDevice

Gas permeable membrane devices are a preferred embodiment of the culturetechniques. Because the membrane is gas permeable, the scale of theculture is determined by the surface area of the membrane and the volumeof media required to grow the cells. Examples of these gas permeabledevices include Hyperstack (Corning) or Grex (Wilson Wolf). One of theuseful features of this type of bioreactor is that the cell cultureprocess is linearly scaleable. As part of the standardization of ourapproach, we have designed versions of bioreactors which slide intostandard CO₂ incubators for use in production suites. In a separateembodiment, we have designed bioreactors which fit into standard stacksfor warm rooms in an automated production facility The bioreactorsintended for automation are made in two sizes: one for growth ofautologous cells for an individual patient and a second larger versionfor the commercial production of allogeneic T cell lines. In an improvedmethod, the cell culture devices are modified for automation into arectangular shape so as to slide into the slots on a standard CO₂incubator. This is a significant advantage as the gas permeable membraneis on the bottom of the flask and hence is blocked if it is sitting onthe shelf. By having the flask be the shelf, there is better airflow tothe membrane. A key attribute is the flanges on the side of the flaskwhich allow them to hold the weight of the bioreactor. In oneembodiment, the bioreactor sets into a stainless steel frame which formsslides into the shelf supports. In another embodiment, they are moldedas part of the plastic. In one of the embodiments, they have thefootprint of an entire shelf. In another embodiment, they comprise ½, ¼or ⅓ Or ⅕ of a shelf. In a preferred embodiment, the incubator is madeby New Brunswick, Form a, ThermoElectron, Nuaire, ESCO. In a preferredembodiment, the incubator can be air or water jacketed or other design.In still another embodiment, they fit within a shelf which is a metalframe or on a flat in a warm room. In another preferred embodiment, theflat is moved and processed robotically. In still another embodiment,the bioreactor fits various commercial processing equipment includingbut not limited to a rocker which brings the cells back into suspensionprior to harvesting.

Each bioreactor is a closed system with access ports to introduce media,components and cells and from which to harvest the cells for freezingand quality control. In a preferred embodiment, these access ports aretubing which ports fluids and cells in and out of the bioreactor the capfor which has integrated solid tubes which reach the bottom of thebioreactor. In an alternative embodiment, the access port is a sterilesheet of rubber through which a needle or other probe can be insertedinto the bioreactor to inject or remove fluid cells or other reagentseither manually or using an automated robot. The devices are bar codedin order to track them so that each patient and each cell line will haveits own dedicated bioreactor. The bioreactor is disposable after it hasbeen used. The bioreactors are also sized to fit standard roboticautomation equipment in automated cell culture including but not limitedto stacks, transporters and rocking agitators. FIG. 31 is an example ofsuch a bioreactor.

Like the bioreactors, commercial multiplexed immune profiling assays arealso designed to enable processing of measuring titers of antibodies andT cell responses to panels of antigens. In order to do this, the ELISPOTassay is used as standard 96 well plate format can be applied. In apreferred embodiment, the AIDELISPOT reader (Autoimmun Diagnostika,Strassberg, Germany) is used to count the spots. Alternatively, the 96well plate is used to input cells into the FACS for the ICS assay. Inany event, each patient has their own dedicated 96 well plate which isbar coded and all plates are disposable. These commercial assays andmanufacturing processes are embodiments of the invention.

E. Examples 1. Example 1 EBV Latent Infection, Lymphomas andNasopharyngial Carcinoma

90% of the world's population has been exposed to EBV (the causativevirus in mononucleosis) as measured by antibodies in the blood. EBVbecomes latent in B cells and shuts off the majority of its proteins butexpress very low levels of latent antigens LMP1, LMP2 and sometimesEBNA-1. These proteins are weakly immunogenic but are required tomaintain the virus even in its latent state. Because they derivepredominantly from B cells, 40% of lymphomas test positive for EBVlatent antigens. Thus, these antigens can serve as targets for thegeneration of CTL responses in adoptive cell therapy. In addition,Nasopharyngial carcinoma also expresses EBV latent antigens as do othertumors (e.g. −10% of gastric cancer). While CTL's have been used totreat EBV lymphomas, the current production methods are time consuming(3-6 months) and cumbersome using B cells transformed with EBV as repeatstimulation. Further, CTL's made in this way to LMP2 produced T cellbatches only half of which had a detectable response to LMP2 postproduction. The inventors believed that the reasons for this were due tothe presence of dominant epitopes from EBV proteins in the EBVtransformed LCL cells which generated CTL's which outgrew the cellsagainst LMP2 50% of the time. In patients with bulky tumors, 52% of thepatients treated with CTL's produced by the traditional process had acomplete response. While the prior art considered all CTL's equivalentregardless of the presence or lack of response to LMP2, the inventorsfelt that this may be one reason for the variable clinical response.Furthermore, the operative model of the invention predicted that thiswas indeed the case. Hence increasing the efficiency of CTL generationto LMP2 may be clinically important.

While other methods of CTL production are also embodied in theinvention, the following methods were used to create the followingexperimental data: 40 ml to 100 ml of peripheral blood was collectedfrom the patients in Vacutainer tubes. Peripheral Blood Mononuclearcells (PBMC's) were isolated by centrifugation on Lymphoprep (Nycomed,Oslo, Norway), resuspended in RPMI 1640 (Gibco, Grand Island, N.Y.)including 2 mM L glutamine, 100 IU/ml penicillin, 100 mgstreptomycin/ml, with 10% Fetal calf serum (FCS) (5×10⁶ cells/ml) andseeded onto 6 well plates (Costar Corp, Cambridge, Mass.) at 10⁷cells/well. After 2 hours at 37 degrees C., nonadherent cells wereremoved and resuspended in FCS with 10% polyethylene glycol (PEG) placedin test tubes, frozen on dry ice and stored in a −80 freezer. Theadherent cells still in the 6 well plate was cultured in RPMI+10% FCSsupplemented with 50 ng of GM-CSF and 1000 U of IL-4 per ml. Half of themedia was replaced with fresh media including the same growth factorsdescribed above on day 2 and day 4. On day 6, the media was completelyreplaced with the media described as well as the addition of 25% volumeof macrophage conditioned medium to stimulate maturation. Macrophageconditioned media was produced by PBMCs adherent to immunoglobulincoated plates (prepared by immunoglobulin in PBS, plating and incubatingat 4 degrees C. overnight) for 24 hours at 37 degrees C. in RPMI 10%FCS, harvesting the supernatant, filtration through a 0.2 mm pore sizemembrane (Acrodisc, Gelman Sciences) and storage at −20 degrees C. forup to 8 weeks before use. Nonadherent cells were harvested 2 days laterand used as a source of dendritic cells. Immunofluorescence stainingwith monoclonal antibodies for surface markers including CD54, CD80,CD83 and CD86 was performed to assure dendritic cell quality (>50% ofcells +).

DC stimulators were preexposed for 2 hours at 37 degrees C. to proteinsat a concentration of μg/ml (50 for peptides) in serum free RPMI 1640supplemented with human 132 microglobulin at 3 μg/ml. They were thenwashed and seeded at 10⁵ cells/2 ml well in RPMI 10% FCS supplementedwith IL-7 5 ng/ml. 2×10⁶ PBMC's were added to each well for a responderto stimulator ratio of 20:1. The cultures were restimulated (and splitinto additional wells, if necessary) on days 14 and 21 with autologouspeptide loaded dendritic cells in RPMI 10% FCS supplemented with IL-2 at20 U/ml.

Release testing of CTL's to be used for treating patients includedviability of >70%, negative culture for bacteria and fungi after 7 days,endotoxin testing less than 5 EU/ml, negative results for Mycoplasma,less than 20% killing of recipient lymphoblasts at a 20:1 ratio in ⁵¹Crrelease assays, less than 2% CD19⁺ B cells, less than 2% CD14⁺ monocytesand HLA identity.

Polyclonal T cell populations were harvested and used as effectors in a5 hr chromium release assay. For the chromium release assays, monolayercultures of fibroblasts established from skin biopsies of CTL donors andexposed to recombinant vaccinia virus (2×10⁶ cells per 9 cm petri dish)Cells harvested 18 hours post transfection and labeled for 1 hour with⁵¹CrO₄, washed three times, and used as targets in a 5 hour chromiumrelease assay. Supernatants from the assay were harvested into 1%formaldehyde before counting on a 7 counter.

a. Experiment 1 The Relative Frequency of T Cells Responding toSubdominant Epitopes is Enhanced when Dendritic Cells or ActivatedMacrophages are Used as Antigen Presenting Cells Instead of LCL

T cell lines were prepared from 10 patients using 3 different antigenpresenting cells as stimulators: EBV transformed lymphoblastoid celllines (LCL) presentation and expansion; Dendritic cell (DC) presentationwith cytokine expansion; INFγ Macrophage (MAC) presentation withcytokine expansion. Each of the 3 were stimulated during antigenpresentation with a mixture of 3 plasmids expressing subdominantepitopes of EBNA-1 (EBNA-1 with deletion of aa 90 to 325), LMP1 (LMP1with deletion of aa 1-43 and aa 260-315) and LMP2 (LMP2A in 2 plasmidsone expressing aa 1-399 and the second plasmid expressing aa 400-497).The T cell lines so generated using the protocol of the invention werethen studied in ⁵¹Cr release assay.

FIGS. 1A and 1B: ⁵¹Cr release at two different effector:target (E/T)ratios, 20:1 and 10:1, respectively, (CTL line: HLA matched fibroblasts)HLA matched fibroblasts pre-pulsed with peptide mixes from the indicatedantigens, CTL's expanded from representative patient using differentindicated APC's to grow CTL line.

Conclusion: Dendritic cells (DC) and Macrophages (MAC) more selectivelystimulate the growth of CTL's to subdominant antigens than EBVtransformed B cells (LCL's).

FIG. 1C: ⁵¹Cr release at three different effector:target (E/T) ratios(CTL's: HLA matched fibroblasts) HLA matched fibroblasts pre-pulsed withpeptide mixes from the indicated antigens CTL's from PBMC's of the samepatient directly after blood harvested (before culture).

Conclusion: Patients have some response to EBV dominant antigens but atsignificantly higher E/T ratios than after T cell culture

FIG. 2: ⁵¹Cr release at 20:1 effector:target (E/T) ratios (CTL Lines:HLA matched fibroblasts) HLA matched fibroblasts pre-pulsed withpeptides representing specific HLA A2 restricted subdominant epitopesfrom LMP2 with CTLs made from the same patient using three differentmethods as above:

Conclusion: Dendritic cells and Macrophages both lead to a response to abroader number of subdominant epitopes & higher levels of CTL activitythan does LCL and the magnitude of the response to different epitopes isdifferent between the Dendritic cells and Macrophages

FIGS. 3A and 3B: ⁵¹Cr release at 20:1 effector:target (E/T) ratios (CTLLines: HLA matched fibroblasts) HLA matched fibroblasts pre-pulsed withpeptides from LMP2: LCL Stimulation in FIG. 3A and DC/MAC stimulation inFIG. 3B.

Conclusion: Dendritic cells and Macrophages lead to CTL Lines in 90% ofthe patients, in contrast to the LCL which only led to 50% of the CTLlines from the patients having a detectable LMP2 response. Therefore,the DC/MAC process is more robust and reproducible for generating Tcells to subdominant antigens and epitopes.

FIG. 3C: % of Viable CD3⁺ Cells which are CD4⁺, CD8⁺ and CD25⁺ in CTLLines grown using Macrophage, Dendritic Cells or LCL cells as AntigenPresenting Cells. Lines were stained with antibodies for CD3, CD4, CD8and CD25 and analyzed by Flow Cytometry.

Conclusion: While all methods establish CD8⁺ cells and not Tregs, theuse of Dendritic cells and macrophages as APC's appears to increase the% of CD4⁺ cells relative to that produced with LCLs.

ELISpot Assays:

Elispot assays were performed to determine the number of T cellsproduced to particular antigens. Elispot γ IFN 96 well polyvinylidenediflouride backed plates (Millipore, Bedford, Mass.) were coated with 15μg/ml of anti-INFγ monoclonal antibody 1-DIK (MABTECH, Stockholm,Sweden). 5×10⁶ PBMCs were added per well with peptide mixes 2 μM eachfrom each of the proteins and incubated overnight at 37 degrees C. 5%CO₂. Cells were discarded and 1 μg/ml biotinylated anti-INFγ monoclonalantibody 7-B6-1 (MABTECH) was incubated 2-4 hours at room temperaturefollowed by streptavidin conjugated alkaline phosphatase (MABTECH) for 2more hours. After a 30 minute reaction with 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium from the alk-phos substrate kit(Bio-Rad Richmond, Calif.), depending upon the number of samples, thespots were counted either using a dissection microscope or on anAIDELISPOT reader (Autoimmun Diagnostika, Strassberg, Germany). Eachspot was a cell reported as spot forming cells (SFC)/10⁵ PBMC's. Inthese assays, the positive control was cells+10 μg/ml PHA and negativecontrol was cells alone without peptide.

FIG. 4: IFN producing cells (SFC's)/105 T cells grown in vitro asmeasured by Elispot

Conclusion: Dendritic cells and Macrophages more selectively expand Tcells to subdominant antigens than LCL

Optimizing Antigens to Establish Responses to Subdominant Epitopes

LMP2 was chosen for optimization as it is a subdominant antigen relativeto EBNA-3 and the majority of EBV subdominant epitopes recognizedappeared to be on this protein as identified using consensus softwareand PBMC testing. In an effort to increase the number of subdominantepitopes recognized, LMP2 was split onto two or more plasmids. Thefollowing plasmids were constructed in the p shuttle or pUC19 plasmidunder control of the CMV promoter and artificial ATG and poly A:

Plasmid 1 LMP2B (aa 1-497)

Plasmid 2 LMP2A 1^(st) exon (aa 1-119) Plasmid 3 LMP2A 2nd exon (aa120-497)

Plasmid 4 LMP2A (aa 120-399) Plasmid 5 LMP2A (aa 400-497) Plasmid 6LMP2A (aa 120-440) Plasmid 7 LMP2A (aa 440-497) Plasmid 8 LMP2A (aa1-399) Plasmid 9 LMP2A (aa 400-497)

All plasmids were generated using standard procedures in SCS 110bacteria strains (Stratagene, La Jolla, Calif.) and purified with Endofree Plasmid Maxi kit (Qiagen, Hilden, Germany). Antigen presentingcells were transfected 24 hours after maturation using the Amaxa DCNucleofection Kit (Amaxa, Koeln, Germany) with 2-20 μg plasmid DNA per10⁶ cells.

T cell immunosubdominant epitopes on LMP2 include but are not limitedto:

LLW 329-337 LLWTLVVLL HLA A 2.01 CLG 426-434 CLGGLLTMV HLA A 2.01 IED200-208 IEDPPFNSL HLA B 40.01 SSC 340-350 SSCSSCPLSKI HLA A11.01 TYG419-427 TYGPVFMCL HLA A24.02 LLS 447-455 LLSAWILTA HLA A2 LTA 453 461LTAGFLIFL HLA A2 FLY 356-364 FLYALALL HLA A2

Peptides for these epitopes were synthesized and used to test CTLresponses in ⁵¹Cr release assays on HLA-A2 expressing fibroblastsisolated from skin of PBMC donors.

FIG. 5: ⁵¹Cr release at 20:1 effector:target (E/T) ratios (CTL Lines:HLA matched fibroblasts) HLA matched fibroblasts pre-pulsed withpeptides from LMP2: CTL Lines established with LMP2 split on 1, 2, 3 or4 plasmids (1P: 497 aa LMP2B; 2P: LMP2A 1^(st) exon (119 aa) on 1plasmid, LMP2A 2^(nd) exon (378 aa); 3P LMP2A 1^(st) exon (119 aa) on 1plasmid, LMP2A (120-399 aa) on plasmid 2 and LMP2A (400-497 aa) on3^(rd) plasmid; 4P: LMP2A 1^(st) exon (119 aa) on 1 plasmid, LMP2A(120-399 aa) on plasmid 2 LMP2A (400-440 aa) on 3^(rd) plasmid LMP2A(440-497 aa) on 4^(th) plasmid; 2P 2: Plasmid 1 (aa 120-440), Plasmid 2(aa 440-497)

Conclusion: Splitting the LMP2 into at least two plasmids one containingaa 400 to 497, the other containing residues before 399 results instronger responses to a greater number of subdominant epitopes

EBNA-1 amino acid 90 to 325 corresponding to Gly Ala repeat domain weredeleted from the EBNA-1 sequence and inserted into the p shuttle plasmidunder the control of the CMV promoter. This sequence was selected fordeletion because it was demonstrated to inhibit peptide processing. Thefollowing HLA-A2 restricted peptide was used to assess response: VLK574-582 HLA A2.

Furthermore, LMP1 sequence was prepared with aa 1-43 deleted (to preventits aggregation/protection from proteosome processing) and 260-315deleted (5 copies of 11 amino acid tandem repeats). These sequences wereconstructed in the p shuttle plasmid under control of the CMV promoterand compared with wild type LMP1. To assess breadth of response, thefollowing HLA-A2 restricted epitopes of LMP1 were prepared and tested:

YLL 125-133 YLLEMLWRL HLA A2 YLQ 159-167 YLQQNWWTL HLA A2 TLL 166-174TLLVDLLWLL HLA A2 LLV 167-175 LLVDLLWLL HLA A2 LLL 92-100 LLLIALWNL HLAA2 RLG 132-140 RLGATIWQL HLA A2

FIGS. 6A and 6B: ⁵¹Cr release at 20:1 effector:target (E/T) ratios (CTLLines: HLA matched fibroblasts) HLA matched fibroblasts pre-pulsed withpeptides from EBNA-1 or LMP1: Comparison of CTL Lines established withEBNA-1 wild type vs. EBNA-1 deleted and LMP1 wild type vs. LMP1 deleted.

Conclusion: Deletion of certain regions of proteins which evade antigenprocessing results in stronger responses to a greater number ofsubdominant epitopes

b. Example 2

In the following experiments, T cells were grown in the same way asoutlined in Example 1 above, with different variations designed toenhance antigen processing to favor T cells reactive with the productionof subdominant epitopes.

FIG. 7: (⁵¹Cr release) Cr release at 20:1 effector:target (E/T) ratios(CTL Lines: HLA matched fibroblasts) HLA matched fibroblasts pre-pulsedwith peptides representing Dominant antigen (EBNA-3A), Subdominantantigen (LMP2) and specific HLA A2 restricted subdominant epitopes fromLMP2 with CTLs made from the same patient using two different methods:one with 100 nM to 300 nM bortezomib added during antigen presentation,one without.

Conclusion: Addition of the Proteosome antagonist bortezomib during CTLantigen presentation modifies antigen processing to generate CTL's to abroader number of subdominant epitopes with a modest decrease in theresponse to dominant epitopes

FIG. 8: (⁵¹Cr release) Cr release at 20:1 effector:target (E/T) ratios(CTL

Lines: HLA matched fibroblasts) HLA matched fibroblasts pre-pulsed withpeptides representing Dominant antigen (EBNA-3A), Subdominant antigen(LMP2) and specific HLA A2 restricted subdominant epitopes from LMP2with CTLs made from the same patient using two different methods: onewith 10 ng/ml Interferon γ (INFγ) added 12 hours before and duringantigen presentation, one without.

Conclusion: Addition of INFγ during CTL antigen presentation modifiesantigen processing to generate CTL's to a broader number of subdominantepitopes with a modest decrease in the response to dominant epitopes.The effect is more dramatic in Macrophages than in dendritic cells.

c. Example 3 Hepatitis and Hepatocellular Carcinoma

A mouse model of chronic hepatitis has been recently developed whichallows the study of the primary and secondary immune response inhepatitis B (Publicover J et al. 2011). This HBVtgRAG mouse is a crossbetween HBV-replication transgenic mice (HBVRpl) in the C57BL/6background for 15 generations which constitutively allows viralreplication and release of virions and RAG-1 deficient mice making themunable to generate T and B cells. When 10⁸ spleenocytes are transferredfrom C57BL/6 mice, the immune system is reconstituted and the primaryimmune response to hepatitis infection is modeled. If one administersthe spleenocytes to young (3-4 week old) mice, the animals (similar toyoung children infected with Hepatitis B) develop chronic Hepatitis Likethe humans, they clear HBc but HBs remains at high levels in the serum(FIG. 1). This model of chronic hepatitis was used in the followingexperiments.

Alanine aminotranferase were measured using ALT-L3K kit (DiagnosticChemicals Ltd) on a Cobas Miras Plus analyzer (Roche diagnostics). HBsAg was measured using ETI-MAX 2 Plus (Diasorin; HBs Antibody wasmeasured using ETI-AB-AUK-PLUS and ABAU standard set (Diasorin). HBcABwas measured using ETI-AB-COREK-PLUS (Diasorin). Assays were read onELx800 (Biotek Instruments) wavelength 450 nm and 630 nm.

FIG. 9: HBVtgRAG mouse model of chronic hepatitis B.

T cells were grown to HBs ex vivo using our protocol and injected bytail vein injection 3-4 weeks post transfer of the originalspleenocytes. A titration was performed from 1×10⁵ to 1×10⁸ cells withplateau achieved at 1×10⁶ cells. As can be seen below, our T cellrebalancing therapy clears chronic hepatitis in the HBVtgRAG model.

FIG. 10: Treatment of HBVtgRAG model with T cells reactive to HBs Aglead to acute inflammation and clearance of the previously chronicinfection.

When IL-4 or IL-21 were used as a supplement to the medium duringantigen presentation, the amount of T cells required to achieve plateaueffect was 5×10⁵ and 7×10⁴, respectively. This number of cells is morethan a log lower than T cells expanded in the standard IL-15 protocoland indicates that polarization during culture towards T follicularhelper cells and TH2 cells is advantageous. On the other hand,polarization with IL-12 increased the number of cells to 1×10⁷ requiredfor plateau and polarization to Treg with IL-2 and rapamycin eliminatesthe response to therapy.

Different Culture conditions result in polarization to different T cellsubsets and different numbers of T cells to reach plateau in clearingHepatitis in the HBVtgRAG mouse

Culture Conditions Number of T cells to Reach Plateau IL-15 1 × 10⁶IL-15 + IL-4 5 × 10⁵ IL-15 + IL-12 1 × 10⁷ IL-15 + IL-21 7 × 10⁴ IL-2 +rapamycin No response

Therefore, polarization of the T cells to different T cell subsets hasimportant therapeutic effects.

To determine the effect of T cell therapy on the frequency of T cells todifferent epitopes, the % of INFγ producing epitopes to differentepitopes was determined by Elispot. At week 6 post transplant,spleenocytes were collected from HBVtgRAG mice who were untreated ortreated with plateau levels of T cells raised to HBs Ag. Thespleenocytes were pulsed with Hepatitis Core Antigen (HBc), HepatitisSurface Antigen (HBs) or two Kb restricted peptides of HBs: ILS or WWL.

HBs Ag 190-197 VWLSVIWM K^(b) HBs Ag 208-215 ILSPFLPL K^(b)

The following results were observed:

FIG. 11: Treatment with HBs T cells rebalances the immune system to anew dominance hierarchy.

As can be seen, T cell lines produced using our protocol generated asignificantly higher T cell response to HBs and a slightly decreasedresponse to HBc. The HBs response was driven predominantly by a de novoresponse to a previously subdominant epitope (WWL). Thus, the dominancehierarchy within HBs was switched by T cell rebalancing therapy as theresponse which exists in untreated animals (albeit weak) is against thedominant epitope (ILS) switching the overall balance of the HBV responsein favor of the previously subdominant HBs antigen relative to thepreviously dominant HBc antigen.

The HBVtgRAG mouse model was also used to investigate whether in vivomethods could also rebalance immunodominance hierarchies. In oneapproach, the inventors injected HBs Antigen in Freund's adjuvant bytail vein injection (IV), intramuscularly (IM) or intraperitoneally(IP). Alternatively, 100μ plasmid DNA was administered per mouse (datanot shown).

FIG. 12: The IM route of administration results in a broader response tosubdominant antigens as dose treatment with a protease inhibitor duringvaccination.

As can be seen, a 4× stronger response is generated by IM administrationrelative to the other two and this is associated with a response to thesubdominant epitope. Furthermore, when this mouse was treated with theproteasome antagonist bortezomib, and the antigen was administered IM,the response to the subdominant epitope was further accentuated.

In a second, set of experiments the response alone and in complexes withtwo HBs reactive murine antibodies of different isotypes (IgG1 vs.IgG2a):

Monoclonal Antibodies to HBs Ag 10-H05 Murine IgG1 HBsAg (Fitzgerald,Concord, Mass.) 10-H05A Murine IgG2a HBsAg (Fitzgerald, Concord, Mass.)

FIG. 13: IgG2a monoclonal antibody but not IgG1 antibody specific forHBs Ag results in a different dominance hierarchy and significantlybetter antigen presentation by the IV route.

When the antigen antibody complex was administered intravenously and IM,the IgG2a complex generated more of a response to the subdominantepitope. Further, this protocol developed a near comparable level ofimmune response by IV administration to that achieved by IMadministration indicating that IV administration of IgG2a immunecomplexes may be able to rebalance the immunodominance hierarchy invivo.

The inventors hypothesize that the likely mechanism for this observationis that IgG2a binds the high affinity FcR which is preferentiallyexpressed on dendritic cells and thus targets the HBs antigen todendritic cells. Additionally, as HBs Ag a determinant first loop (aa124-147) is the major epitope for recognition by neutralizing antibodiesand the ILS epitope is further away, it could be that antigen processingfor ILS is enhanced.

Clinical Trial of T Cell Rebalancing Therapy in HBV AssociatedHepatocellular Carcinoma

5 patients with HBV and HCC who were HLA-A*0201 were immune profiled fortheir Humoral and cellular immune response. Pretreatment clinicallaboratory testing demonstrated high titers of antibody to hepatitis Bcore antigen (HBc Ag) but not to hepatitis B surface antigen (HBs Ag)(FIG. 10).

PBMC's were induced into dendritic cells, pulsed with hepatitis B coreantigen (HBc Ag) and hepatitis B surface antigen (HBs Ag) and used togrow CTL's from PBMC's which were tested by Elispot for INFγ.

FIG. 14: Number of Spot forming cells in 10⁵ PBMCs from Patient 3.

By frequency of IFN γ producing T cells on Elispot a hierarchy wasobserved: no-antigen (10 sfc)<HBsAg(15 sfc)<HBcAg(45 sfc). Thisindicates that Hepatitis B surface antigen is subdominant relative toHepatitis core antigen. Additionally, 3 peptides of HBs (FLL, GLS andILS) were studied:

HBs Ag 20-28 FLLTRILTI HLA-A*201 HBs Ag 185-194 GLSPTVWLSV HLA-A*201 HBsAg 208-216 ILSPFLPLL HLA-A*201

The FLL peptide appeared to be slightly dominant in what little responseto HBs was detectable but, because of the weak response, the dominancehierarchy was confirmed by ICS assays:

Intracellular Cytokine Staining (ICS)

5×10⁵ CTL were resuspended and incubated for an hour in 100 μ1 of 1×PBS1% FCS with peptide (10⁻⁵ to 10⁻⁹ M final concentration) Golgi Plug (BDBiosciences, San Diego, Calif.) was then added, cells were incubated at37 degrees C. 5% CO₂ for 5 hours, pelleted, washed in 200 μl PBS 1% FCSand stained for surface antigens (CD4 fluorescein isothiocyanate and CD8allophycocyanin (Pharmingen, Becton Dickinson) 30 minutes at 4 degreesC. Following resuspension in fixation and permeabilization solution,cells were stained with anti human INFγ phycoerythrin (Pharmingen, BD)1/20 dilution on ice for 30 minutes, washed once and resuspended in PBS1% FCS and analyzed on FACS Canto (Becton Dickinson)

FIG. 15: ICS Analysis of response to HBs epitopes.

Although the responses detectable by ICS weren't strong, the FLL epitopedid again appear dominant relative to the other two epitopes.

5×10⁶ PBMC's were isolated from each patient and T cells were grown asper our protocol using HBs 15 mer peptides mixes as the stimulatingantigen with the peptide with the exclusion of peptide 15-30 (toeliminate FLL). IL-4+IL-15 were used during the expansion. 1×10¹⁰ Tcells were obtained and frozen with an aliquot taken for Elispottesting.

FIG. 16: HBs Cells are the majority of the T cells which areadministered to the patient and these respond to previously subdominantantigens.

As can be seen, the CTL line generated was almost exclusively reactiveto the previously subdominant HBs, a response which was primarily drivenby the previously subdominant epitopes. CTL's were dosed by IV Infusionat 5×10⁷ to 1×10⁸ cells/m² and the patients were monitored.

FIG. 17: The patient has an acute flair and then clears the hepatitis.

After developing acute hepatitis, the patient resolved and completelycleared the hepatitis. On day 14, PBMC's were again collected and aprofile of the cellular immune response was re-determined.

FIG. 18: The patient's immunodominance hierarchy has been rebalanced tothe previously subdominant antigen (HBs) and the previously subdominantepitopes on that antigen.

The immunodominance hierarchy has been successfully rebalanced by the Tcells grown by our process. In addition to clearing the chronichepatitis, the patient also had a complete response to hisHepatocellular carcinoma.

FIG. 19: The T cells completely resolved the patient's Hepatocellularcarcinoma.

d. Example 4 Systematic Method for Treating Cancer

The clinical diagnosis and therapy of cancer involves the biopsy andimaging of the tumor to determine its tissue of origin, differentiationand extent of local and systemic metastasis. While diagnostics togenetic defects in oncogenes or tumor suppressor genes and assays todetermine sensitivity to chemotherapeutic or biologic agents issometimes performed, generally patients are treated with a combinationof surgery, chemotherapy and radiation depending upon their specificcancer and stage. Similarly, while the immune system in various patientshas been studied using different techniques, this information has notbeen used in the clinical management of the patients. With the advent oftheir cellular therapy to rebalance the immune system, the inventors hadto establish the use of immune profiling in the management of the cancerpatient. The inventors have developed a standardized immune profilingmethodology which is used to select antigens which are subdominant inthat patient and can be used to grow T cells in vitro which can bereinfused into the patient to rebalance the immune response to a tumor.Following therapy, the patient is reprofiled to determine if the therapysuccessfully rebalanced the immune response. Such a therapeutic methodis novel and can result in clinical response and enhanced survival. Thesame approach can be used infectious diseases and autoimmunity and organtransplantation.

The first step is to identify which tumor associated antigens arepresent on the patient's tumor. This is generally done byimmunohistochemistry on a biopsy from the patient's tumor. The panelantigens tested will depend on the type of tumor. For example, inmelanoma antigens included NY-ESO-1, SSX-2, Melan A, gp100, MAGE A4,MAGE A1, Tyrosinase and would be supplemented as new tumor associatedantigens were described. Not all tumors will express all of the antigensin the panel. For the antigens, which the patients tumor has, a baselineimmune profile to the different antigens is determined using thesystematic profiling for humoral and cellular immune response describedabove. Elisa is used to determine antibody titer to each antigen in theserum and the % of T cells responding to the antigen is determined byINFγ ICS or Elispot. In this way, a base line profile is determined.

FIG. 20: INFγ ICS to a Panel of Tumor Antigens in Patient 1

In Patient 1, the patient's tumor had NYESO-1, SSX-2, Melan A, and MAGEA4. ICS demonstrated a strong response to MAGE A4 but modest/no responseto NYESO-1, SSX-2 and Melan A. The NYESO-, SSX-2 and Melan A antigensare thus chosen to grow and expand CTL in the in vitro culture using thefollowing protocol:

5×10⁶ PBMC's are isolated from the blood. Monocyte-derived dendriticcells are generated in vitro from peripheral blood mononuclear cells(PBMCs) from a patient by plating of PBMCs for 2 hours in a tissueculture flask to permit adherence of monocytes. At this point thenonadherent cells are removed and frozen at −80 to later serve as asource of T cells. Treatment of the adherent monocytes with interleukin4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF)leads to differentiation to immature dendritic cells (iDCs) in about aweek. Subsequent treatment with tumor necrosis factor (TNF) furtherdifferentiates the iDCs into mature dendritic cells. These cells arethen separated into 3 separate flasks (or however many antigens to whichone desires to grow T cells) in RPMI 1640 media supplemented with 45%Click's medium (Irvine Scientific, Santa Ana, Calif.), 2 mM Glutamax 1and 5% human serum. Each flask is pulsed with one of the 3 plasmids (orpepmix) containing the coding sequence for the antigen of interest (inthis patient 1 containing NY-ESO-1,1 containing SSX-2 and the thirdcontaining Melan A). The cells are stored at 37 degrees C. for 2 hours.In the meantime, PBMC's are thawed and added to the pulsed dendriticcells at a 1:20 to 1:100 PBMC to Dendritic cell ratio and incubated at37 degrees C. for 18 hours. The three flasks of cells are then pooledand resuspended in the same media, containing IL-15 (5 ng/ml) togenerate in vitro expansion of the T cells which have recognized theantigen. A Grex gas permeable bioreactor is used (Wilson WolfManufacturing Minneapolis, Minn.) obviating the need to change media andenabling exponential growth kinetics. Generally 3×10⁸ to 1.5×10¹⁰ cellsare obtained within 3 to 6 weeks enough cells to be administered to apatient.

T Cells Grow in Gas Permeable Bioreactor

T cells grow as a layer on the gas permeable membrane for excellent gasexchange and have a volume of media sufficient to grow to the requireddensity.

At the end of in vitro culture, T cells are assayed in the ICS assaywith the same antigens: NY-ESO-1, SSX-2, Melan A, gp100, MAGE A4, MAGEA1, Tyrosinase.

FIG. 21: ICS Assay Patient 1

Based upon the assay, the % of the cells responding to each subdominantantigen/epitope is determined in this case, NY-ESO-1, SSX-2 and Melan A.The total number of cells responding to each subdominant antigen orepitope can be calculated using this % and the number of CTLs in theculture. In the case of patient 1, 61.4% of the cells were responding toNY-ESO-1 (FIG. 21).

Based upon this, a known dose of CTLs responsive to the subdominantantigens can be administered to the patient. CTL's were dosed at 5×10⁶to 2×10⁸ cells/m².

2-3 weeks after infusion into the patient, PBMC's were again collectedand a profile of the cellular immune response was re-determined by ICS:

FIG. 22: Cellular Immune Profiling by ICS post therapy-Patient 1

FIG. 23: Humoral Immune Profiling by ELISA-Patient 1 (Reciprocal titersof the humoral immune response are plotted below).

As can be seen from the Cellular and Humoral Profiles from the ImmuneHierarchy Assays, the immunodominance hierarchy was rebalanced to favorthe previously subdominant antigens.

Clinical Findings:

Clinically, the patient was a 52 year old male who was diagnosed withStage 1V metastatic melanoma. He had failed to respond to a combinationregimen of Dacarbazine (DTIC) and Temodar (Temolzolomide) chemotherapydrugs in combination with IL-2.

Two years post T cell therapy, the patient is alive and has undergone acomplete response. As can be seen in the Chest CT Scan the metastasis inthe lung completely resolved within 6 months of Immune rebalancingTherapy and has remained stable.

FIG. 24: CT Scan Pre and Post T Cell Therapy

e. Example 5 T Cell Therapy of Lymphoma

150 adult patients with relapsed aggressive Non Hodgkins lymphoma wererandomized into 3 arms: A: Rituxan+CHOP; B: Testing for EBV LMP1 & LMP2antigens in tumor, followed by EBV LMP1 & LMP2 T cell rebalancing and C:Pan lymphoma: No assay for antigens; Therapy with T cells grown torespond to EBV LMP1, LMP2, surviving, MAGE A3.40% of lymphoma biopsiestest positive for EBV LMP2, 50% test positive for surviving and 15% testpositive for MAGE A3. Standard of Care R-CHOP regimen was used and 5×10⁷to 2×10⁸ T cells were dosed per m².

FIG. 25: Progression Free Survival

As can be seen, T cells responsive to the tumor provide superiorresponse rates to the current standard of care. While failures occur inyear 1, post year 1 the CTL maintains patients in remission. This isevidence of a properly functioning immune system post rebalancing.Finally, while initially the EBV LMP T cells provide a better response,by year 3 the Progression Free Survival has approached that of the Panlymphoma product. Furthermore, lymphoma today is a relapsing remittingdisease with patients generally relapsing within 18 months to 2 years.CTL rebalancing therapy changes this course: once a patient's immunesystem is rebalanced, the patient develops a memory response whichmaintains a long term remission. Thus, unlike other therapies oflymphoma, T cell therapy provides a durable remission.

FIG. 26: T Cell Therapy Changes the Natural History of Disease

f. Example 6 T Cell Therapy of Autoimmune Diseases

The collagen induced arthritis model (CIA) is a model of RheumatoidArthritis (RA) that can be induced by immunization with heterologouscollagen II (CII) in DBA/1 mice.

DBA/1 male 6-8 week old mice were obtained from Jackson Laboratories(Bar Harbor, Me.). 100μ of bovine CII (Chondrex, Redmond, Wash.)emulsified in CFA containing 4 mg/ml M. tuberculosis (Chondrex) wereinjected subcutaneously in the tail. By week 5 post injection, 80-100%of untreated mice showed fully developed disease.

T cells were grown to the following peptides using the protocolsdescribed for the in vitro growth of T cells.

Collagen Type II

263-270 immunodominant peptide used to stimulate the growth of Treg(IL-2+ rapamycin) 286-300 subdominant peptide used to stimulate growthof T cells (IL-15)

5×10⁶ cells were administered to each animal on Day 20 post inductionalone or in a combination of equal parts. See FIG. 27. CD25 is a markerfor Treg cells. Control animals received PBS. See FIG. 28.

Mice were scored for clinical disease three times per week using a scoreof 0-3 for each limb for a maximum total score of 12 possible: 0-1Normal; 1 mild redness or swelling in single digits; 2 significantswelling of ankle or wrist with erythema; 3 severe swelling and erythemaof multiple joints. The percent of animals with arthritic lesions in thegroup represent incidence of arthritis. Average clinical score in thegroup reflects severity of the disease. See FIG. 28 and FIG. 29.

Histopathology of Joint

FIG. 30A shows a normal rat, FIG. 30B shows a rat immunized with humanproteoglycan and FIG. 30C shows a rat treated with T cells.

As can be seen, the T cell therapy significantly decreased theincidence, severity and amount of inflammation in the joint of animals.There appeared to be a synergistic effect in rebalancing the immuneresponse between T cells raised to subdominant epitopes and Treg grownto the dominant epitope.

1. A method for producing a culture expanded, cancer subdominant antigenresponsive T cell population, the method comprising: contacting human Tcells ex vivo with a plurality of fragments of a subdominant antigenfrom a cancer; contacting the T cells with agents that promote antigenresponsive T cell growth and expansion, comprising growth factors,hormones, immune cells and cytokines; and enriching for the populationof culture expanded subdominant antigen responsive T cells, wherein, theT cell population is capable of altering an immunodominance hierarchy ina patient having the cancer.
 2. The method of claim 1, wherein the humanT cells are obtained from a tumor.
 3. The method of claim 1, wherein thehuman T cells are obtained from peripheral blood mononuclear cells(PBMC).
 4. The method of claim 1, further comprising the step ofidentifying a subdominant antigen in the cancer, prior to contactinghuman T cells with the fragments of the subdominant antigen.
 5. Themethod of claim 1, further comprising the step of identifying asubdominant antigen in a patient having the cancer, prior to contactinghuman T cells with the fragments of the subdominant antigen.
 6. Themethod of claim 1, wherein the fragments in the aggregate represent afull length form of the subdominant antigen.
 7. The method of claim 1,wherein the plurality of fragments do not include regions of the cancersubdominant antigen that evade antigen processing.
 8. The method ofclaim 1, wherein the culture expanded T cell population comprisesmultiple subpopulations, wherein each subpopulation is responsive to anindividual fragment of the subdominant antigen.
 9. The method of claim1, wherein the T cells are contacted with more than one subdominantantigen in the cancer.
 10. The method of claim 9, wherein the cultureexpanded T cell population is responsive to multiple cancer subdominantantigens.
 11. The method of claim 1, wherein the cancer is selected fromthe group consisting of: EBV malignancies, a carcinoma, a lymphoma, ablastoma, a sarcoma, a melanoma, a neuroblastoma, and a hepatocellularcarcinoma.
 12. The method of claim 1, wherein the subdominant antigen isselected from the group consisting of: NY-ESO1, SSX-2, MAGE-A4, MAGE A1,Melan A, gp100, cancer related viral antigens, LMP1, LMP2, EBNA-1, HSV,HPV E6, HPV E7, HB core antigens, HB surface antigens, Hepatitis Cantigens, HIV antigens, HTLV antigens and CMVpp65 antigens.
 13. Themethod of claim 1, wherein the subdominant antigen is a peptide, aprotein or a nucleic acid.
 14. The method of claim 3, further comprisingseparating the PBMC into antigen presenting cells and T cells prior tocontacting the T cells with the fragments of the subdominant antigen,wherein the T cells comprise naïve T cells, and wherein the antigenpresenting cells comprise monocytes, dendritic cells (DC) andmacrophages.
 15. The method of claim 1, wherein the T cells do notcomprise T cells responsive to a dominant antigen in the cancer.
 16. Themethod of claim 1, wherein the immune cells that promote antigenresponsive T cell growth and expansion include antigen presenting cellsstimulated by the subdominant antigen, wherein the antigen presentingcells are selected from monocyte derived dendritic cells, macrophagesand EBV immortalized B cells.
 17. The method of claim 1, wherein thecytokines that promote antigen responsive T cell growth and expansionare selected from the group consisting of: IL-2, IL-4, IL-6, IL-7,IL-12, IL-15, IL-21, and any combination thereof.
 18. The method ofclaim 17, wherein the cytokines comprise IL-15, IL-2 or both.
 19. Themethod of claim 18, wherein the cytokines further comprise IL-7, IL-21and IL-12.
 20. The method of claim 1, wherein the cytokines comprise acombination of any two or more cytokines selected from the groupconsisting of: IL-2, IL-7, IL-15 and IL-21.
 21. The method of claim 20,wherein, IL-2 is used at a concentration ranging between 2 Units(U)/mland 1000 U/ml, IL-7 is used at a concentration ranging between 1 ng/mland 150 ng/ml, IL-15 is used at a concentration ranging between 1 ng/mland 150 ng/ml, or IL-21 is used at a concentration ranging between 1ng/ml and 150 ng/ml.
 22. The method of claim 1, the method furthercomprising the step of: confirming the presence of antigen responsive Tcells by detecting in the culture expanded T cell population, cells withone or more T cell activation markers.
 23. The method of claim 1,wherein the culture expanded T cell population comprises CD8+ cytotoxicT cells responsive to the subdominant antigen.
 24. The method of claim1, wherein the culture expanded T cell population comprises CD4+ Tcells, TH1 and TH2 polarized CD4+ T cells responsive to the subdominantantigen.
 25. The method of claim 1, wherein the culture expanded T cellpopulation comprises at least 5% cytotoxic T cells responsive to thecancer subdominant antigen.
 26. The method of claim 1, wherein theculture expanded T cell population comprises cytotoxic T cellsubpopulations responsive to multiple cancer subdominant antigens.